Angiogenesis is a biological process to provide a new blood vessel to organs or tissues. Particularly, new blood capillaries are generated from an old microvessel, which are growing to become blood vessels. Angiogenesis, as a normal physiological process, is observed only in limited cases in a human body, for example during the development of fetus and embryo, during the maturation of uterus, during the proliferation of placenta, during the formation of corpus luteum, and for the healing of a wound. And even for such cases, angiogenesis is accurately regulated and stopped when it finishes its job. Angiogenesis is regulated strictly by an angiogenesis regulating factor (Folkman, J., Nature Med., 1: 27-31, 1995) and the phenotype of angiogenesis seems to differ from the balance between up-regulation of an angiogenesis stimulating factor and down-regulation of an angiogenesis-inhibiting factor.
Although the angiogenesis process is very complicated and sophisticated, it can be outlined as follows. First, a stimulus for angiogenesis is transmitted to an old blood vessel, resulting in the expansion of the blood vessel and the increase of membrane permeability. Second, fibrin is coming out of the expanded blood vessel and then accumulated in cytosol around the blood vessel. Third, an enzyme to decompose the basal membrane of the old blood vessel is activated. Fourth, the basal membrane is destroyed so that endothelial cells come out of the blood vessel and proliferate in cytosol around and then move again. Lastly, those endothelial cells are lined up to form a vessel, resulting in a new blood vessel.
The diseases related to angiogenesis are exemplified by inflammatory diseases including arthritis, ophthalmic diseases including diabetic retinopathy, dermatological diseases including psoriasis and cancer which is the most representative angiogenesis related disease among all other diseases (Folkman, J., Nature Med., 1:27-31, 1995). In particular, angiogenesis is essential for the growth of a primary tumor and a metastatic tumor (Folkman, J., New Engl. J. Med., 285:1182-1186, 1971; Folkman, J., J. Biol. Chem., 267:10931-10934, 1992). That means a tumor cannot be growing when angiogenesis is inhibited or stopped, indicating that inhibition of angiogenesis might be an effective way for long term treatment of tumors.
Therefore, it is urgently required to develop a novel angiogenesis inhibitor to inhibit or to stop angiogenesis and further to provide the inhibitor to a user by moderate prices resulted from effective mass-production of the angiogenesis inhibitor.
Angiogenesis inhibitor is regarded as one of the most favorable treatment methods for cancer because it targets blood vessels supplying nutrients to a tumor, instead of attacking directly cancer cells, which means it reduces resistance against medicine. Various inhibitors to inhibit angiogenesis in a tumor, such as natural inhibitors found in vivo, synthetic inhibitors, integrin inhibitors, signal transduction inhibitors and proteolysis inhibitors, have been verified to be effective and are on clinical test (Brower, V., Nat. Biotechnol., 17:963-8, 1999; Carmeliet, P. and Jain, R. K., Nature, 407:249-57, 2000). Among those inhibitors on the clinical test, LK6, LK7 and LK8, which are the 36th, 37th and 38th kringle subunits of Apo(a) of human apolipoprotein, stated in international patent publication WO 01/19868, have been known to have angiogenesis inhibiting activity along with activities of growth inhibition and migration inhibition of epithelial cells. Especially, LK8 protein shows the highest activity. In order to provide moderate prices for the production of LK8 protein for the examination of the effect of the protein, for clinical test and for commercial use, the development of a method for mass-production of LK8 protein through the culture of recombinant strain transformed by LK8 gene is essential.
Thus, the present inventors have studied on the functions of LK8 protein and the method for effective commercial use of the protein. At last, the present inventors prepared recombinant Saccharomyces cerevisiae by transforming yeast Saccharomyces cerevisiae with plasmid vector containing LK8 gene or inserting LK8 gene into host chromosome, and then selected strains mass-producing LK8 protein therefrom. The present inventors also determined the optimal culture condition for the growth of the strains and finally completed this invention by confirming that high quality LK8 protein could be mass-produced under said condition.